Preoperative prolapse phenotype is predictive of surgical outcome with minimally invasive sacrocolpopexy.

BACKGROUND: The gold-standard treatment for advanced pelvic organ prolapse is sacrocolpopexy. However, the preoperative features of prolapse that predict optimal outcomes are unknown. OBJECTIVE: This study aimed to develop a clinical prediction model that uses preoperative scores on the Pelvic Organ Prolapse Quantification examination to predict outcomes after minimally invasive sacrocolpopexy for stages 2, 3, and 4 uterovaginal prolapse and vaginal vault prolapse. STUDY DESIGN: A 2-institution database of pre- and postoperative variables from 881 cases of minimally invasive sacrocolpopexy was analyzed. Data from patients were analyzed in the following 4 groups: stage 2 uterovaginal prolapse, stage 3 to 4 uterovaginal prolapse, stage 2 vaginal vault prolapse, and stage 3 to 4 vaginal vault prolapse. Unsupervised machine learning was used to identify clusters and investigate associations between clusters and outcome. The k-means clustering analysis was performed with preoperative Pelvic Organ Prolapse Quantification points and stratified by previous hysterectomy status. The "optimal" surgical outcome was defined as postoperative Pelvic Organ Prolapse Quantification stage <2. Demographic variables were compared by cluster with Student t and chi-square tests. Odds ratios were calculated to determine whether clusters could predict the outcome. Age at surgery, body mass index, and previous prolapse surgery were used for adjusted odds ratios. RESULTS: Five statistically distinct prolapse clusters (phenotypes C, A, A>P, P, and P>A) were found. These phenotypes reflected the predominant region of prolapse (apical, anterior, or posterior) and whether support was preserved in the nonpredominant region. Phenotype A (anterior compartment prolapse predominant, posterior support preserved) was found in all 4 groups of patients and was considered the reference in the analysis. In 111 patients with stage 2 uterovaginal prolapse, phenotypes A and A>P (greater anterior prolapse than posterior prolapse) were found, and patients with phenotype A were more likely than those with phenotype A>P to have an optimal surgical outcome. In 401 patients with stage 3 to 4 uterovaginal prolapse, phenotypes C (apical compartment predominant, prolapse in all compartments), A, and A>P were found, and patients with phenotype A>P were more likely than those with phenotype A to have ideal surgical outcome. In 72 patients with stage 2 vaginal vault prolapse, phenotypes A, A>P, and P (posterior compartment predominant, anterior support preserved) were found, and those with phenotype A>P were less likely to have an ideal outcome than patients with phenotype A. In 297 patients with stage 3 to 4 vaginal vault prolapse, phenotypes C, A, and P>A (prolapse greater in posterior than in anterior compartment) were found, but there were no significant differences in rate of ideal outcome between phenotypes. CONCLUSION: Five anatomic phenotypes based on preoperative Pelvic Organ Prolapse Quantification scores were present in patients with stages 2 and 3 to 4 uterovaginal prolapse and vaginal vault prolapse. These phenotypes are predictive of surgical outcome after minimally invasive sacrocolpopexy. Further work needs to confirm the presence and predictive nature of these phenotypes. In addition, whether the phenotypes represent a progression of prolapse or discrete prolapse presentations resulting from different anatomic and life course risk profiles is unknown. These phenotypes may be useful in surgical counseling and planning.

Islet primary cilia motility controls insulin secretion.

Primary cilia are specialized cell-surface organelles that mediate sensory perception and, in contrast to motile cilia and flagella, are thought to lack motility function. Here, we show that primary cilia in human and mouse pancreatic islets exhibit movement that is required for glucose-dependent insulin secretion. Islet primary cilia contain motor proteins conserved from those found in classic motile cilia, and their three-dimensional motion is dynein-driven and dependent on adenosine 5'-triphosphate and glucose metabolism. Inhibition of cilia motion blocks beta cell calcium influx and insulin secretion. Human beta cells have enriched ciliary gene expression, and motile cilia genes are altered in type 2 diabetes. Our findings redefine primary cilia as dynamic structures having both sensory and motile function and establish that pancreatic islet cilia movement plays a regulatory role in insulin secretion.

Generation of ciliary beating by steady dynein activity: the effects of inter-filament coupling in multi-filament models.

The structure of the axoneme in motile cilia and flagella is emerging with increasing detail from high-resolution imaging, but the mechanism by which the axoneme creates oscillatory, propulsive motion remains mysterious. It has recently been proposed that this motion may be caused by a dynamic 'flutter' instability that can occur under steady dynein loading, and not by switching or modulation of dynein motor activity (as commonly assumed). In the current work, we have built an improved multi-filament mathematical model of the axoneme and implemented it as a system of discrete equations using the finite-element method. The eigenvalues and eigenvectors of this model predict the emergence of oscillatory, wave-like solutions in the absence of dynein regulation and specify the associated frequencies and waveforms of beating. Time-domain simulations with this model illustrate the behaviour predicted by the system's eigenvalues. This model and analysis allow us to efficiently explore the potential effects of difficult to measure biophysical parameters, such as elasticity of radial spokes and inter-doublet links, on the ciliary waveform. These results support the idea that dynamic instability without dynamic dynein regulation is a plausible and robust mechanism for generating ciliary beating.

Quantifying Ciliary Dynamics during Assembly Reveals Stepwise Waveform Maturation in Airway Cells.

Motile cilia are essential for clearance of particulates and pathogens from airways. For effective transport, ciliary motor proteins and axonemal structures interact to generate the rhythmic, propulsive bending, but the mechanisms that produce a dynamic waveform remain incompletely understood. Biomechanical measures of human ciliary motion and their relationships to ciliary assembly are needed to illuminate the biophysics of normal ciliary function and to quantify dysfunction in ciliopathies. To these ends, we analyzed ciliary motion by high-speed video microscopy of ciliated cells sampled from human lung airways compared with primary culture cells that undergo ciliogenesis in vitro. Quantitative assessment of waveform parameters showed variations in waveform shape between individual cilia; however, general trends in waveform parameters emerged, associated with progression of cilia length and stage of differentiation. When cilia emerged from cultured cells, beat frequency was initially elevated, then fell and remained stable as cilia lengthened. In contrast, the average bending amplitude and the ability to generate force gradually increased and eventually approached values observed in ex vivo samples. Dynein arm motor proteins DNAH5, DNAH9, DNAH11, and DNAH6 were localized within specific regions of the axoneme in the ex vivo cells; however, distinct stages of in vitro waveform development identified by biomechanical features were associated with the progressive movement of dyneins to the appropriate proximal or distal sections of the cilium. These observations suggest that the stepwise variation in waveform development during ciliogenesis is dependent on cilia length and potentially on outer dynein arm assembly.

Finite element models of flagella with sliding radial spokes and interdoublet links exhibit propagating waves under steady dynein loading.

It remains unclear how flagella generate propulsive, oscillatory waveforms. While it is well known that dynein motors, in combination with passive cytoskeletal elements, drive the bending of the axoneme by applying shearing forces and bending moments to microtubule doublets, the origin of rhythmicity is still mysterious. Most conceptual models of flagellar oscillation involve dynein regulation or switching, so that dynein activity first on one side of the axoneme, then the other, drives bending. In contrast, a "viscoelastic flutter" mechanism has recently been proposed, based on a dynamic structural instability. Simple mathematical models of coupled elastic beams in viscous fluid, subjected to steady, axially distributed, dynein forces of sufficient magnitude, can exhibit oscillatory motion without any switching or dynamic regulation. Here we introduce more realistic finite element (FE) models of 6-doublet and 9-doublet flagella, with radial spokes and interdoublet links that slide along the central pair or corresponding doublet. These models demonstrate the viscoelastic flutter mechanism. Above a critical force threshold, these models exhibit an abrupt onset of propulsive, wavelike oscillations typical of flutter instability. Changes in the magnitude and spatial distribution of steady dynein force, or to viscous resistance, lead to behavior qualitatively consistent with experimental observations. This study demonstrates the ability of FE models to simulate nonlinear interactions between axonemal components during flagellar beating, and supports the plausibility of viscoelastic flutter as a mechanism of flagellar oscillation.

Flexural Rigidity and Shear Stiffness of Flagella Estimated from Induced Bends and Counterbends.

Motile cilia and flagella are whiplike cellular organelles that bend actively to propel cells or move fluid in passages such as airways, brain ventricles, and the oviduct. Efficient motile function of cilia and flagella depends on coordinated interactions between active forces from an array of motor proteins and passive mechanical resistance from the complex cytoskeletal structure (the axoneme). However, details of this coordination, including axonemal mechanics, remain unclear. We investigated two major mechanical parameters, flexural rigidity and interdoublet shear stiffness, of the flagellar axoneme in the unicellular alga Chlamydomonas reinhardtii. Combining experiment, theory, and finite element models, we demonstrate that the apparent flexural rigidity of the axoneme depends on both the intrinsic flexural rigidity (EI) and the elastic resistance to interdoublet sliding (shear stiffness, ks). We estimated the average intrinsic flexural rigidity and interdoublet shear stiffness of wild-type Chlamydomonas flagella in vivo, rendered immotile by vanadate, to be EI = 840 ± 280 pN⋅µm(2) and ks = 79.6 ± 10.5 pN/rad, respectively. The corresponding values for the pf3; cnk11-6 double mutant, which lacks the nexin-dynein regulatory complex (N-DRC), were EI = 1011 ± 183 pN·µm(2) and ks = 39.3 ± 6.0 pN/rad under the same conditions. Finally, in the pf13A mutant, which lacks outer dynein arms and inner dynein arm c, the estimates were EI = 777 ± 184 pN·µm(2) and ks = 43.3 ± 7.7 pN/rad. In the two mutant strains, the flexural rigidity is not significantly different from wild-type (p > 0.05), but the lack of N-DRC (in pf3; cnk11-6) or dynein arms (in pf13A) significantly reduces interdoublet shear stiffness. These differences may represent the contributions of the N-DRCs (∼40 pN/rad) and residual dynein interactions (∼35 pN/rad) to interdoublet sliding resistance in these immobilized Chlamydomonas flagella.

Dynein-deficient flagella respond to increased viscosity with contrasting changes in power and recovery strokes.

Changes in the flagellar waveform in response to increased viscosity were investigated in uniflagellate mutants of Chlamydomonas reinhardtii. We hypothesized that the waveforms of mutants lacking different dynein arms would change in different ways as viscosity was increased, and that these variations would illuminate the feedback pathways from force to dynein activity. Previous studies have investigated the effects of viscosity on cell body motion, propulsive force, and power in different mutants, but the effect on waveform has not yet been fully characterized. Beat frequency decreases with viscosity in wild-type uniflagellate (uni1) cells, and outer dynein arm deficient (oda2) mutants. In contrast, the inner dynein arm mutant ida1 (lacking I1/f) maintains beat frequency at high viscosity but alters its flagellar waveform more than either wild-type or oda2. The ida1 waveform is narrower than wild-type, primarily due to an abbreviated recovery stroke; this difference is amplified at high viscosity. The oda2 mutant in contrast, maintains a consistent waveform at high and low viscosity with a slightly longer power stroke than wild-type. Analysis of the delays and shear displacements between bends suggest that direct force feedback in the outer dynein arm system may initiate switching of dynein activity. In contrast, I1/f dynein appears to delay switching, most markedly at the initiation of the power stroke, possibly by controlling inter-doublet separation.

Equations of interdoublet separation during flagella motion reveal mechanisms of wave propagation and instability.

The motion of flagella and cilia arises from the coordinated activity of dynein motor protein molecules arrayed along microtubule doublets that span the length of axoneme (the flagellar cytoskeleton). Dynein activity causes relative sliding between the doublets, which generates propulsive bending of the flagellum. The mechanism of dynein coordination remains incompletely understood, although it has been the focus of many studies, both theoretical and experimental. In one leading hypothesis, known as the geometric clutch (GC) model, local dynein activity is thought to be controlled by interdoublet separation. The GC model has been implemented as a numerical simulation in which the behavior of a discrete set of rigid links in viscous fluid, driven by active elements, was approximated using a simplified time-marching scheme. A continuum mechanical model and associated partial differential equations of the GC model have remained lacking. Such equations would provide insight into the underlying biophysics, enable mathematical analysis of the behavior, and facilitate rigorous comparison to other models. In this article, the equations of motion for the flagellum and its doublets are derived from mechanical equilibrium principles and simple constitutive models. These equations are analyzed to reveal mechanisms of wave propagation and instability in the GC model. With parameter values in the range expected for Chlamydomonas flagella, solutions to the fully nonlinear equations closely resemble observed waveforms. These results support the ability of the GC hypothesis to explain dynein coordination in flagella and provide a mathematical foundation for comparison to other leading models.

Whole-exome capture and sequencing identifies HEATR2 mutation as a cause of primary ciliary dyskinesia.

Motile cilia are essential components of the mucociliary escalator and are central to respiratory-tract host defenses. Abnormalities in these evolutionarily conserved organelles cause primary ciliary dyskinesia (PCD). Despite recent strides characterizing the ciliome and sensory ciliopathies through exploration of the phenotype-genotype associations in model organisms, the genetic bases of most cases of PCD remain elusive. We identified nine related subjects with PCD from geographically dispersed Amish communities and performed exome sequencing of two affected individuals and their unaffected parents. A single autosomal-recessive nonsynonymous missense mutation was identified in HEATR2, an uncharacterized gene that belongs to a family not previously associated with ciliary assembly or function. Airway epithelial cells isolated from PCD-affected individuals had markedly reduced HEATR2 levels, absent dynein arms, and loss of ciliary beating. MicroRNA-mediated silencing of the orthologous gene in Chlamydomonas reinhardtii resulted in absent outer dynein arms, reduced flagellar beat frequency, and decreased cell velocity. These findings were recapitulated by small hairpin RNA-mediated knockdown of HEATR2 in airway epithelial cells from unaffected donors. Moreover, immunohistochemistry studies in human airway epithelial cells showed that HEATR2 was localized to the cytoplasm and not in cilia, which suggests a role in either dynein arm transport or assembly. The identification of HEATR2 contributes to the growing number of genes associated with PCD identified in both individuals and model organisms and shows that exome sequencing in family studies facilitates the discovery of novel disease-causing gene mutations.